PROJECT SUMMARY/ABSTRACT Idiopathic pulmonary fibrosis (IPF) is a chronic disease of the lung characterized by the differentiation of resident fibroblasts into contractile myofibroblasts that deposit excessive extracellular matrix (ECM). There are no effective treatments for IPF, and the median survival time after diagnosis is approximately 3 years. Increased matrix deposition is a hallmark of IPF that increases the stiffness of lung tissue, thereby encouraging fibroblast differentiation into myofibroblasts and furthering disease progression. Increased matrix deposition concomitantly increases the degree of confinement experienced by cells, yet the role of confinement in fibroblast differentiation is unknown. Mesenchymal stem cell (MSC) injection is currently being targeted as a potential therapeutic for IPF in clinical trials. However, some studies indicate that MSC therapy worsens outcome, yielding conflicting results. Studies have suggested that protective effects are due to MSC secreted factors, which are often collected from MSCs cultured in 2D on standard tissue culture plastic. It has been shown that manipulating the MSC microenvironment alters the MSC secretome, yet the effect of confinement on the MSC secretome is unknown. We hypothesize that a) increasing confinement experienced by fibroblasts will encourage their differentiation into myofibroblasts, and b) increasing confinement experienced by MSCs will increase their protective effects on fibroblasts, inhibiting myofibroblast differentiation. To investigate this hypothesis, we propose two Specific Aims: 1) Evaluate the effect of matrix composition and degree of confinement on fibroblast to myofibroblast differentiation, and the role cell mechanics play in this process, and 2) Evaluate the effect of MSC secreted factors in various degrees of confinement on differentiation of fibroblasts to myofibroblasts and matrix deposition by fibroblasts and myofibroblasts. For aim 1) fibroblasts will be cultured within confining devices and their differentiation into myofibroblasts will be characterized via ?-SMA immunofluorescence staining and gene and protein expression analysis of characteristic myofibroblast markers. Traction forces and chromosome condensation will be investigated as potential players in the differentiation mechanism. For aim 2) MSCs will be cultured within confining devices and their secreted factors collected. These secreted factors will be applied to fibroblasts, and fibroblast to myofiroblast differentiation will again be characterized. Extracellular vesicles will be investigated as a potential contributor to the therapeutic effects of MSC secretions. Thus, we aim to determine the role of confinement and ligand presentation in lung fibroblast and myofibroblast mechanics, and their response to MSC secretions. Successful completion of these aims will improve understanding of IPF progression and improve methods of MSC culture for use in IPF treatments. Broadly, this research will enhance understanding of human pulmonary biology, advance translational research, and reduce human disease. This training fellowship will be facilitated by the University of Maryland Fischell Department of Bioengineering.